Endoplasmic reticulum (ER) stress may also
promote a senescent-like response. The
accumulation of unfolded proteins in the ER triggers a stress-signaling pathway
that can result in cell cycle arrest mediated by p27 (Han et al. 2013) and the p53/47 isoform (Bourougaa et al. 2010). Furthermore,
ER stress has also been shown to induce an inflammatory response via NFkB
activation (Garg et al. 2012) and
induce cytokines such as MCP-1, IL-6 and IL-8 (Schroder, 2008), which are
capable of attracting and activating immune cells (Sagiv and Krizhanovsky, 2013).
ER stress has also been shown to promote cell survival, another feature of cell
senescence (Raciti et al. 2012). Interestingly, a senescent state via
activation of ER stress-dependent p21 signaling has been reported in proximal
tubular epithelial cells, triggered by receptors for advanced glycation
end-products (RAGE) (Liu et al. 2014). Although, ER stress-induced senescence has
the potential induce an immunogenic phenotype in the absence of DNA damage, a full
evaluation of the phenotype is required to determine if this is so.

Metabolic stress, defined here as a combination
of aerobic glycolysis and mitochondria dysfunction can potentially trigger a senescent
state. All organisms that use aerobic
glycolysis form reactive acyclic α-oxoaldehydes
(e.g. methylglyoxal and glyoxal) spontaneously from triosephosphates and by a
wide variety of other routes (Thornalley, 2009). These dicarbonyl compounds are highly
reactive and damage proteins through non-enzymatic modification producing a
wide variety of covalent adducts (AGEs).
Elevated levels of methylglyoxal and glyoxal are known to be cytotoxic
and although the mechanism of action remains imprecisely defined, it can be
blocked by ROS scavengers, suggesting that oxidative stress mediates at least
some of the deleterious effects (Shangari and O’Brian, 2004).

Cytosolic and mitochondrial protection from dicarbonly
damage is primarily mediated through the action of the glyoxalase system that
consists of two enzymes, glyoxalase I and II.
However, in cultures of WI38 fibroblasts a significant reduction in the
activity of glyoxalase-I occurs with serial passage (Ahmed et al. 2010). Treatment of cultures
of ASF2 human adult dermal fibroblasts with micro or millimolar concentrations of
glyoxal or methylglyoxal renders them senescent within 72 hours. This was defined by the presence of typical
senescent morphology, irreversible growth arrest and increased SA-β-Gal
activity (Sejersen & Rattan, 2009). Further
studies (Larsen et al. 2012) extended
these observations to immortalized human mesenchymal stem cells (MSCs) and demonstrated
that treatment with physiologically reflective (Han et al. 2007) concentrations of glyoxal for 72 hours led to
senescence without significant cell death (although massive cell death occurred
at higher glyoxal concentrations). Elevated
levels of SA-β-Gal, p16 and DNA damage (as measured by
COMET) accompanied the growth arrest. Interestingly,
a profound reduction in the ability of these senescent MSCs to differentiate
into functional osteoblasts (as determined by alkaline phosphatase and
mineralization assays) was also observed.
Given the imbalances in glucose metabolism that accompany mammalian
ageing (and diabetes), the authors proposed that this type of metabolic stress
might underlie age-related changes in bone function. Unfortunately, no markers of immunogenic
conversion have yet been measured in this system and whilst the presence of DNA
damage could indicate the likelihood of a secretory response, this cannot be
assumed. Thus, the propensity of
senescence human MSCs to be cleared by the immune system remains unknown and is
of considerable physiological significance.

TGFβ-induced senescence: A growing body of evidence suggests that the members of the transforming growth factor beta (TGF- β) family can induce a senescence-like state. Experimentally, senescence has been predominantly, but not exclusively, characterized by the presence of senescence-associated beta galactosidase (SA-β-Gal) staining and the up-regulation of cyclin dependent kinase inhibitors (CDKi) (see below). Human prostate basal cells treated with TGF-β1/2/3 show increased SA-β-Gal activity, which is associated with the flattened, and enlarged cell morphology typical of adherent senescent cells in vitro (Untergasser et al. 2003). Similarly TGF-β1 has been reported to induce a senescent state in bone marrow mesenchymal stem cells as a result of increased mitochondria ROS production (Wu et al. 2014). These cells also showed SA-β-Gal staining and an increased expression of p16. Yu et al. (2010) demonstrated that TGF-β2 could induce a senescent-like state in human trabecular meshwork cells. Again, this was associated with SA-β-Gal staining, increased levels of p16 at both the message and protein level and a reduction in the level of pRB protein. No impact on p21 mRNA or protein expression was observed in response to TGF-β2 exposure. Other groups have also reported a role for TGF-β signaling in inducing a senescent state (Senturk et al. 2010, Minagawa et al. 2011, Acosta et al. 2013).

It is generally accepted that SA-β-Gal staining should be used in conjunction with several other senescent markers, as it does not appear to detect senescent cells specifically (Severino et al, 2000). However, other than the expression of CDKi, it appears that the phenotypes of cells induced to enter senescence by exposure to TGF-βs have been poorly characterized, especially in regard to immunogenic conversion. Some cell types that become senescent via this route may be cleared by the immune system in a manner analogous to those undergoing developmentally programmed senescence. Others may not and this area represents a fruitful field for further investigation.

Developmentally programmed senescence: Cells sharing features of senescence have been reported within the mesonephros and the endolymphatic sac of the inner ear in human and mouse embryos; as well as the neural roof plate and apical ectodermal ridge in rodents (Munoz-Espin et al. 2013, Storer et al. 2013). The authors hypothesize that this “developmental senescence” (DS) is a programmed part of normal embryonic development. DS was demonstrated experimentally by the presence of SA-β-Gal activity and senescence associated heterochromatin (Munoz-Espin et al. 2013). These cells seem to lack detectable DNA damage and appear to have become senescent independent of p53 and p16 and have gene expression patterns that significantly overlap with those of IMR90 fibroblasts in a state of oncogene-induced senescence. Arrest in this instance is dependent instead upon p21, regulated via the TGF-β/SMAD and PI3K/FOXO pathways (thus showing some affinity with other TGF-β induced senescent states). Interestingly, DS cells are removed during normal embryonic development by macrophages in a manner related to immune clearance of senescent cells in the mature organism (or by apoptosis should senescence fail) contributing to the formation of normal tissue architecture. Thus, the long-recognized distinction between programmed cell death in development and apoptosis in the mature organism appears to be mirrored in DS. Given that the expression of p21 in developing embryos is often attributed to ‘terminal differentiation’ (Vasey et al. 2011), it will be interesting to determine how many of these p21 positive cells are senescent cells and have undergone immunogenic conversion.Taken from:Cellular Senescence: From Growth Arrest to Immunogenic Conversion

In addition to secreting soluble factors for the
attraction of immune cells, senescent cells can also become immunogenic through
the up-regulation of ligands that can specifically be recognized by immune
cells. While research into the
recognition and interaction of immune cells with senescent cells is at its
infancy, a number of studies have reported the up-regulation of the Natural
Killer Group 2D (NKG2D) ligands in senescent cells that can be recognized by
receptors on Natural Killer (NK) cells and CD8+ T-cells. Since NKG2D ligands are not widely expressed
on healthy cells, this would allow for specific recognition, interaction and
elimination of senescent cells by immune cells.
As with the senescent secretome, this response is likely not exclusive
to cell senescence as the same mechanism functions in immunosurveillance of
tumour cells (López-Soto et al. 2014). The human NKG2D ligands primarily consist of
MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. The transcriptional up-regulation of MICA and
ULBP2 during cell senescence have been reported in senescent activated hepatic
stellate cells, replicative senescent fibroblasts and HUVECs, etoposide-induced
senescent fibroblasts, fusion-induced senescent fibroblasts and
chemotherapy-induced senescent multiple myeloma cells (Krizhanovsky, et al. 2008, Kim
et al. 2008 Chuprin et al. 2013, Soriani et al. 2014, Lackner et al, 2014). In addition to MICA and ULBP2, microarray
analysis of replicative senescent fibroblasts demonstrated an increase in the
expression of ULBP1 (2.75 fold) compared to growing cells, in addition to the
up-regulation of HLA-E (2 fold) (Lackner et
al. 2014). HLA-E is a non-classical
MHC class I molecule that plays a role in cell recognition by NK cells. However,
replicative senescent vascular smooth muscle cells do not appear to up-regulate
MICA, ULBP2 or ULBP1, at least not greater than 2 fold as assessed by microarray
analysis (Burton et al. 2009). Therefore, it should not be assumed that all
senescent cell types up regulate NKG2D ligands and this should be evaluated in
underexplored senescent cell types. Mechanisms involved in the interaction of
senescent cells with T-cells is less understood, but it appears that major
histocompatibility complex class II (MHCII) expression is required for killing
of pre-malignant senescent hepatocytes by T-cells (Kang et al. 2011). Mice with
liver specific MHCII deficiency resulted in impaired immunosurveillance of
senescent cells.

At the mechanistic level, little
is currently known about the regulation of NKG2D ligand expression in senescent
cells. Nonetheless, some extrapolation
from others models is possible. For
example, MICA and MICB have been reported to be regulated by endogenous miRNAs
in tumours and as a result of infection with cytomegalovirus (Stern-Ginossar et al. 2008). Since miRNAs appear to play a role in
regulating cellular senescence (Feliciano et
al. 2011, Liu et al. 2012
Benhamad et al. 2012) and their
expression is altered in response to DNA damage (Dolezalova et al. 2012, Wang and Taniguchi, 2013), it
is possible that changes in miRNA expression also regulate the expression of
immune ligands in senescent cells.

Soriani et
al demonstrated that the up-regulation of MICA in senescent multiple
myeloma cells was dependent upon the DDR (Soriani
et al. 2014). In other systems, NKG2D ligands have also
been shown to be up-regulated in response to DNA damage and Ras activation via
ATM and ATR (Gasser et al. 2005,
Cerboni et al. 2014). Inhibition of the ATM or ATR pathways
prevented the up-regulation of immune ligands.

It is also possible that the up-regulation of
immune ligands on senescent cells is mediated via the secretory response. In addition to activating and attracting
immune cells, the senescent secretome may serve to up-regulate immune ligands in
an autocrine or paracrine manner. It has
been shown for example, that TNFα can
up-regulate MICA on human endothelial cells and that the addition of exogenous
MICA seems to induce senescence in HUVECs (Lin et al. 2011), but the extent to which this occurs under more
physiologically reflective situations remains unclear.

Immune ligands can also be up-regulated in
response to various other forms of cell stress such as heat shock, metabolic
stress and endoplasmic reticulum (ER) stress (Cerwenka, 2009, Valés-Gómez et al. 2008). Thus, as with the secretory response, mechanisms
exists that can up-regulate immune ligands independent of DNA damage. Given that this is an important aspect of
senescent cell clearance and the number of cell types in which the up-regulation
of immune ligands has been shown is limited, a more detailed study of this
aspect of immunogenic conversion seems warranted.

While senescent cells are likely eliminated by
the immune system during normal physiological processes, it has been speculated
that the accumulation of senescent cells with age could be due to inefficient
elimination by an ageing immune system (Burton, 2009). In fact, immune cells may themselves undergo
cellular senescence, a process that requires further investigations (Effros et al. 2005, Rajagopalan et al. 2012). As such, induction of cell
senescence in immune cells may represent one aspect of immunosenescence, the
gradual deterioration of the immune system, which consequently leads to
impaired immunosurveillance of non-immune senescent cells. It can be speculated that impaired
immunosurveillance may result from altered expression of surface receptors on
immune cells that impair recognition and interaction with target senescent
cells (and cancer cells). In addition,
it is possible that aged or senescent immune cells do not respond as efficiently
to chemoattractants secreted by senescent cells. In order to understand the mechanisms
associated with age-related changes resulting in impaired immunosurveillance of
senescent cells, we must first fully understand the normal processes governing
immune clearance of senescent cells.
However, evaluating the hypothesis that aged or senescent immune cells display
a reduced capacity to target senescent cells and the physiological impact of
this decline can still be assessed. If
this were indeed found to be the case, the rejuvenation of an ageing immune
system would represent an attractive approach for promoting health span.

Taken from:Cellular Senescence: From Growth Arrest to Immunogenic ConversionIn order to develop senotherapeutic drugs (targeting cellular senescence), it is important to understand the molecular mechanisms governing the pro-survival phenotype of senescent cells.Senescent cells are frequently referred to as
‘apoptosis resistant’. This apparent resistance
to an apoptotic stimulus in vitro was
originally reported by Wang (1995) who observed that late passage (58
population doubling) WI38 fibroblasts were resistant to death caused by serum
withdrawal compared to WI38 cultures at less than 15 or approximately 38
population doublings. All of these human
cell populations were dramatically more resistant to death by growth factor
deprivation than Swiss 3T3 fibroblasts.
This death resistant phenotype was linked to maintenance of Bcl2 protein
levels in senescent WI38 cells.
Subsequent studies extended the resistance phenotype to treatment with
both UV light (120mJ) and staurosporin (35nM) and linked it to reduced
expression of caspase 3 (Marcotte et al.
2004). Subsequent work (Ryu et al. 2007) using human dermal
fibroblasts confirmed resistance to staurosporin-induced cell death and
demonstrated significant resistance to thapsigargin (up to 700nM). The enhanced survival of senescent dermal
fibroblasts under these conditions was attributed to a failure to down regulate
Bcl2 under conditions of cellular stress.

It has been proposed that resistance to
apoptotic cell death is a feature of the senescent phenotype that may promote
their persistence in vivo, therebyfavoring immune clearance over cell
death. However, key questions around this phenotypic aspect remain and may be
summarized as (i) what are the primary molecular players driving apoptosis
resistance in senescent human dermal and lung fibroblasts? (ii) is this
phenomenon a general one across tissues and between species?

It is possible that the pro-survival response observed
in fibroblasts normally facilitates DNA repair, but is maintained when
persistent DNA damage activates the senescent program. For example, when low levels of DSBs are
present, ATM and ATR can result in ERK/NFkB pro-survival signaling (Khalil et al. 2010, Hawkins et al. 2011, Janssens and Tschopp, 2006)
that has been associated with the induction of senescent cells by various
triggers. Paradoxically ATM-deficient
human fibroblasts are significantly more resistant to cell death triggered by
exposure to doxorubicin or low dose ionizing radiation than wild type controls
(Park et al. 2012). However, the population doublings levels of
the wild type and mutant cultures were not reported. If significantly different, this has the
potential to confound studies of this type (since normal fibroblast cultures
are mixtures of senescent and proliferating cells, the proportions of which
alter as the culture is passaged).

In addition to activating cell cycle arrest in
response to DNA damage, the p53/p21 pathway can also initiate a pro-survival
response. In some studies, p21 has been
shown to play a role in cell survival through its cytoplasmic localization,
rather than its nuclear localization associated with cell cycle arrest (Gartel
and Tyner, 2002, Piccolo and Crispi 2012, Kreis et al. 2014). Interestingly, p21 has been reported to be a negative
regulator of p53-mediated apoptosis (Gartel and Tyner, 2002), a known response
reported in senescent fibroblasts (Seluanov et
al. 2001). p21 has also been
reported to promote cell survival in response to oxidative stress by
integrating the DDR with endoplasmic reticulum (ER) stress signaling (Vitiello et al. 2009). However, the up-regulation of p21 may also be
required for cells to enter and maintain quiescence (Perucca et al. 2009), suggesting a pro-survival
response may occur independent of DNA damage, but dependent upon growth
state.

Autophagy is another feature of senescent cells which
can also be initiated by DNA damage and promote cell survival (Rodriguez-Rocha et al. 2011, Singh et al. 2012). Autophagy
promotes cell survival by the degradation of damaged cellular components (Codogno
and Meijer, 2005), probably as a result of elevated ROS (Scherz-Shouval and
Elazar, 2011) in the case of cell senescence.
Interestingly, there is crosstalk between autophagy and apoptosis
pathways (Zhou et al. 2011, Xu et al. 2013, Lindqvist and Vaux, 2014),
with particular emphasis on the anti-apoptotic Bcl2 protein family.

It has long been recognized that cytokines and
their binding proteins can act to modulate cell survival (Lotem and Sachs, 1999). Given the altered secretory phenotype of some
senescent cells, it would be unsurprising if this did not contribute to altered
death dynamics, but the mechanisms by which this could occur are potentially
highly complex. For example Interleukin-6
(secreted by senescent cells) has been shown to promote cell survival in
transformed cells (Biroccio et al.
2013), and its secretion by cancer-associated fibroblasts protects luminal
breast cancer cells from tamoxifen treatment (Sun et al. 2014). Whilst inhibition
of insulin-like growth factor-1 (IGF-1) has been shown to induce apoptosis in senescent
fibroblasts (Luo et al. 2014), the
alteration of IGF-1 binding proteins are just as likely to influence cell
survival. For example, insulin-like growth factor binding protein 3
(IGFBP-3) is both transcriptionally up-regulated and secreted in elevated amounts
by senescent human fibroblasts (Hampel et
al. 2005). IGFBP-3 triggers enhance
apoptotic cell death in tumor cells when internalized and translocated to the
nucleus, where it targets intracellular regulators of apoptosis (Hampel et al. 2005). Endocytotic uptake of
IGFBP-3 in senescent human fibroblasts did not occur. This has the potential to
render them apoptosis resistant and capable of promoting apoptosis in cells
nearby. It could be speculated that in a
microenvironment characterized by high cell turnover, both senescent and
precancerous cells could be in close proximity. Elevated local IGFBP-3
generated by senescent cells could thus act as a paracrine tumour suppression
mechanism. This idea remains untested.

It seems doubtful that global apoptosis
resistance is a general feature of senescent cells. For example, early work by one of us (RGAF)
failed to show any elevation in spontaneous apoptosis rates in HUVECs cultured
to senescence (although baseline apoptosis rates as measured by TUNEL were significantly
higher than those seen in fibroblasts) (Kalashnik et al. 2000). Later studies (Hoffman
et al. 2001) demonstrated that late
passage HUVECs were more sensitive to apoptosis induced by oxidized LDL or TNFα compared to early passage cells. Jeon and Boo (2013) have recently shown that up-regulation
of the Fas receptor at both the mRNA and protein level in senescent HUVECs
probably underlies their enhanced potential to undergo programmed cell death. Perhaps most compellingly, Hample et al. (2004) demonstrated in parallel
culture experiments that whilst senescent human dermal fibroblasts were more resistant
to cell death induced by exposure to ceramide than early passage cells,
senescent HUVECs were significantly more apoptosis prone.

Interestingly, Crescenzi et al. (2011) have recently shown that induction of premature
senescence in human cancer cell lines also induces Fas expression, and
concomitant susceptibility to Fas-induced apoptosis. Fibroblasts rendered senescent by serial
passage are also susceptible to Fas-mediated killing (Tepper et al. 2000). Thus it is possible that at senescence, human
cell types differ in their resistance to apoptosis induced by stressors, but
show a common susceptibility to Fas/TNFαmediated killing.If immunogenic
conversion were a key hallmark of senescence, then this would seem
plausible. It does however require
significant additional experimental study.

As with the secretory response, it should not be
assumed that an “apoptosis resistant” phenotype is conserved across
species. For example Mayogora et al. (2004) demonstrated that cultures
of cardiac fibroblasts from Sprague-Dawley rats were more resistant to apoptosis
induced by serum withdrawal or staurosporin, than dermal fibroblast cultures initiated
from the same animals. Dermal
fibroblasts from this species apparently lacked Bcl2 protein as measured by
Western blot (although it remained readily detectable in cardiac
fibroblasts). This is a clear species
difference and suggests that researchers working in other systems should not
assume that the features observed in human cells are duplicated across the
animal kingdom.